The focus of this proposal is the mechanism of mammalian translation initiation, which requires at least 9 initiation factors (eIFs), and is a target for multiple regulatory pathways. It occurs in two stages: formation of a 48S initiation complex at the initiation codon of mRNA and its joining with a 60S ribosomal subunit. First, 43S preinitiation complex comprising a 40S ribosomal subunit, a ternary complex of eIF2, initiator tRNA and GTP, and eIFs 3, 1 and 1A attaches to the capped 5'-proximal region of mRNA in a step that involves unwinding of its secondary structure by eIFs 4A, 4B and 4F, and then scans to the initiation codon. After initiation codon recognition and formation of the 48S complex with established codon-anticodon base-pairing, eIF5 and eIF5B promote hydrolysis of eIF2-bound GTP, displacement of eIFs from the 40S subunit and joining of a 60S subunit. The proposed studies will be based on the approach of in vitro reconstitution of all stages of protein synthesis (initiation, elongation, termination and ribosomal recycling) from individual purified translational components. In Aim 1, we will investigate the mechanistic aspects of entry of eIF4F-bound capped mRNAs into the mRNA-binding cleft of the 40S subunit during attachment of 43S complexes by determining the position of eIF4E in ribosomal initiation complexes, identifying the first position in a capped mRNA at which an AUG codon can interact productively with initiator tRNA, and following the fate of the cap-eIF4E- eIF4G-eIF3-40S chain of interactions during the transition from ribosomal attachment to scanning. In Aim 2, we propose to investigate the network of DEAD/DExH-box proteins that have currently been implicated in initiation (e.g. eIF4A, Ded1, DHX29 etc.) by characterizing their relative individual activities at distinct stages of initiation (ribosomal attachment and scanning) and their potential synergy in promoting ribosomal scanning through stable mRNA secondary structures. We also propose to develop fast kinetics techniques to measure kinetic parameters of scanning and to determine how they differ depending on the helicases and other factors involved. Aim 3 will be devoted to investigation of the mechanism of action of various physiologically important translation regulators that have been implicated in protein synthesis by studies in vivo. In Aim 4, we will characterize mechanisms of post-recycling regulation of initiation, focusing on two processes, preferential shunting of recycled 40S subunits back to the 5'-end of the same mRNA, and reinitiation after translation of short open reading frames. PUBLIC HEALTH RELEVANCE: Protein synthesis is of central importance in cell metabolism, and its complex initiation stage is a target for multiple regulatory pathways that integrate it with developmental processes and with changes in the cellular environment. Accordingly, defects in the initiation process can cause severe inherited diseases such as hereditary thrombocythemia and congenital erythroid aplasia, and aberrant cell growth and proliferation, for example in tumors. These studies will determine the molecular basis for key events in translation initiation, and its regulation by trans-acting factors, which is a prerequisite for the development of rational therapies to treat such diseases. )